Volume 36, Issue 5

In recent years the growing interest in quantitative applications of the polymerase chain reaction (PCR) has favoured the development of a large number of assay procedures suitable for this purpose. In this paper we review some basic principles of quantitative PCR and in particular the role of reference materials and calibrators and the different strategies adopted for nucleic acid quantification. We focus on two methodological approaches for quantitative PCR in this review: competitive PCR and real-time quantitative PCR based on the use of fluorogenic probes. The first is one of the most common methods of quantitative PCR and we discuss the structure of the competitors and the various assay procedures. The second section is dedicated to a recent promising technology for quantitative PCR in which the use of fluorogenic probes and dedicated instrumentation allows the development of homogeneous methods. Assay performance of these methods in terms of practicability and reliability indicates that these kinds of technologies will have a widespread use in the clinical laboratory in the near future.

Endemic nephropathy is a chronic renal disease with a high prevalence in a geographically limited area of Croatia. It has also been recorded in some parts of Bosnia, Serbia, Bulgaria and Romania. Despite numerous studies conducted to date, the etiology of this disease has not been clarified. Pathological studies of the kidney in the early stage of endemic nephropathy have shown renal tubules to be the primary sites of the pathologic process with an interstitial tissue reaction, whereas glomerular alterations are of a secondary character. Tubulointerstitial lesions can thus account for the symptoms of the disease, i.e. tubular proteinuria and reduced urine concentration capacity and urine acidification. Also, an increased incidence of malignant tumours of the urinary tract was found in the same geographic area.

Single-strand conformation polymorphism analysis was used to screen for mutations in exon 3 of the low density lipoprotein receptor gene in a group of 218 unrelated patients with severe hypercholesterolemia (low density lipoprotein cholesterol > 6.7 mmol/l) living in the Cologne area of Germany. Including the complementary primers the fragment studied had a length of 176 bp. An abnormal single-strand conformation polymorphism pattern was observed in eight patients, four of whom had an identical abnormal fragment pattern indicating that five different mutations were present. By direct DNA sequencing, the underlying mutations could be confirmed (Cys 54 →Tyr, Trp 66 →Gly, Glu 80 →Lys, 2 bp insertion (AT between codon 44 and 45, 9 bp deletion (codons 65 to 67)). The analysis of the pathogenicity indicates that all mutations were causative for the low density lipoprotein cholesterol elevation. The Trp 66 →Gly and Glu 80 →Lys mutations were previously described in a French-Canadian population and in an English population, respectively. The 2 bp insertion was detected in four unrelated patients and is one of the most frequent mutations detected up to now in the German population.

Cytoplasmic heart-type fatty acid-binding protein has recently gained much attention in clinical diagnosis as a very early marker of acute myocardial infarction. Immunoassays have been developed for determination of this protein in plasma and urine samples. In the present study it is shown that those types of fatty acid-binding proteins which are abundant in tissues other than heart and muscle do not interfere with immunochemical determination of heart-type fatty acid-binding protein. To provide sufficient protein of consistent quality as standard in these immunoassays, human heart-type fatty acid-binding protein was cloned, expressd in Escherichia coli and purified to homogeneity. For quantitation of the recombinant protein its extinction coefficient was determined. Comparison of the recombinant and tissue-derived proteins by a variety of methods revealed both proteins to show similar kinetic as well as equilibrium constants with respect to two monoclonal antibodies currently applied in immunochemical detection of heart-type fatty acid-binding protein. Both preparations were indistiguishable in sandwich-ELISA and immunosensor measurements. A high stability of the recombinant protein was proven by ELISA measurements during storage and several freeze and thaw cycles. Thus, recombinant and tissue-derived heart-type fatty acid-binding proteins are immunochemically equivalent. The recombinant human heart-type fatty acid-binding protein is now available as standard for immunoassays.

We studied transketolase activity of red blood cell hemolysates, and Na + , K + , ATPase activity and sialic acid concentration in red blood cell membranes from 52 patients with rheumatoid arthritis and 24 control subjects. Decreased red blood cell membrane Na + , K + , ATPase activity and sialic acid concentration and decreased transketolase in red blood cell hemolysates were observed in rheumatoid arthritis patients compared with control subjects (p < 0.001). Erythrocyte sedimentation rate and C-reactive protein values were increased in rheumatoid arthritis patients compared with control subjects (p < 0.0001). Significant correlations between sialic acid and Na + , K + , ATPase (r = 0.65, p < 0.001) and between sialic acid and transketolase (r = 0.58, p < 0.001) were observed. Erythrocyte sedimentation rate and C-reactive protein levels did not correlate with Na + , K + , ATPase activity or with sialic acid or transketolase in rheumatoid arthritis patients. These data show that decreases in Na + , K + , ATPase, and transketolase activities and sialic acid concentration are present in rheumatoid arthritis patients, and that the decrease in Na + , K + , ATPase and transketolase activities in rheumatoid arthritis might be due to decreased sialic acid.

Alkaline protease from Pseudomonas aeruginosa (E.C. 3.4.24.40), also called pseudomonal serralysin, plays an important role in the infection bacterial process. The alkaline protease concentration and activity in the culture supernatants of 23 Pseudomonas aeruginosa strains from patients suffering from cystic fibrosis were determined by using an ELISA procedure and a specific enzymatic activity test. No correlation was demonstrated between the activity and the concentration of alkaline protease. This could be explained by the presence of an inactive precursor and a partial hydrolysis of the enzyme, confirmed by SDS-PAGE and Western blotting in the supernatants. The activity test is the only one allowing a quantification of the presence of an active form of alkaline protease.

We prepared a candidate primary reference material for the forthcoming international standardisation of β-N-terminal glycated hemoglobin A measurements. It consists of well-defined mixtures of purified β-N-terminal glycated hemoglobin A and non-glycated hemoglobin A. First, β-N-terminal glycated hemoglobin A and non-glycated hemoglobin A were isolated, purified to homogeneity, and characterised. The techniques used were cation exchange and affinity chromatography for the purification, and high performance liquid chromatography, capillary isoelectric focusing, electrospray ionisation mass spectrometry, and peptide mapping for the characterisation. Hemoglobins from blood of healthy, non-diabetic volunteers were obtained with a purity of >99.5% for non-glycated hemoglobin A and of >98.5% for β-N-terminal glycated hemoglobin A. However, results from peptide mapping indicate that the β-N-terminal glycated hemoglobin A preparations still contain some non-β-N-terminal glycated hemoglobins, co-eluting with β-N-terminal glycated hemoglobin A. The exact content of β-N-terminal glycated hemoglobin A in these preparations could be determined by a procedure consisting of standard addition, enzymatic cleavage and quantification of the resulting β-Nterminal peptides to be in the range from 95–97.5%. Since the β-N-terminal glycated hemoglobin A and non-glycated hemoglobin A content could be exactly determined in the materials prepared, mixtures of both components could be successfully used to calibrate the candidate reference methods.

Purpose: The aim of the study was to evaluate prospectively urinary α 1 -microglobulin as a marker of proximal tubular damage following acute pyelonephritis and outflow disease of the upper urinary tract in a urological population with minimal exclusion criteria. We also measured the urinary γ-glutamyltransferase activity, urinary albumin, urinary and serum creatinine, serum IgA and serum α 1 -microglobulin. Patients and methods: We studied 483 urological patients (age: 1 to 92 years, 297 men, 186 women) excluding patients receiving nephrotoxic drugs, or suffering from type 1 diabetes or renal diseases. There were 141 patients with urinary tract infection but no fever, 36 patients with high fever of non-renal origin, 51 patients with acute pyelonephritis and 156 patients with outflow disease of the upper tract, and 99 patients were incluced in the reference population. Results: For acute pyelonephritis, vesico-ureteral reflux, and ureteral obstruction, urinary α 1 -microglobulin had a sensitivity of 94%, 90% and 63% respectively and a specificity of 67%, 77% and 76%. The area under the curve of the receiver operator characteristic curve was significantly (p < 0.001) higher for urinary α 1 -microglobulin than for albumin or γ-glutamyltransferase activity. Unexpected positive results were found in acute prostatitis. The urinary α 1 -microglobulin was the only parameter which differentiated between acute prostatitis and pyelonephritis (p < 0.001). Creatinine clearance or age had little and gender had no influence on the urinary excretion of α 1 -microglobulin. Urine production rate significantly increases the urinary α 1 -microglobulin/creatinine ratio. Conclusion: Our results suggest that the urinary α 1 -microglobulin/creatinine ratio is a diagnostically useful marker of tubular damage in acute pyelonephritis and vesico-ureteral reflux in the urological population. Following renal colic and chronic ureteral obstruction, a significant increase in urinary α 1 -microglobulin excretion was observed.

Lipoprotein(a) is a unique lipoprotein with atherothrombogenic properties. Although its blood concentration is mainly genetically determined, various factors exist which may cause variability. These may influence the clinical use of the results. We studied lipoprotein(a) biological variation by a rate nephelometric assay over a period of two years in a population of healthy fertile women. The study was performed in 12 volunteers, healthy subjects with various lipoprotein( a) concentrations, by monthly determinations during one year and a single determination one year later, together with measurements of total, high density lipoprotein and high density lipoprotein2 cholesterol, triglycerides and apolipoproteins A1 and B. The intra-individual variability of lipoprotein(a) ranged between 4 to 20%, with three subjects showing a coefficient of biological variation higher than 15 %. In absolute terms, the difference between two determinations could represent 0.44 g/l or 50 % of the mean value. This study suggests that physiological lipoprotein( a) variations should be taken into account for clinical purposes, especially in patients in need of thorough risk evaluation.

The effects of the time of the day and breakfast on the outcome of interleukin-6 and its soluble receptor in serum were studied on 22 subjectively healthy volunteers. We collected blood specimens at 8.00 a.m. after an overnight fast, at 9.30 a.m. after usual breakfast, and at 11.30 a.m. A second group of volunteers (n = 20) was studied during the evening. We observed a significant decrease in the concentration of interleukin-6 after breakfast. This suggests that for assay of this analyte it is preferable to collect specimens after overnight fast. No changes were observed in the concentrations of interleukin-6 soluble receptor. Weak, but significant negative correlations were found between the age of the volunteers and the concentrations of interleukin-6 soluble receptor and between age and the ratio interleukin-6 soluble receptor/interleukin-6. The concentrations of interleukin-6 were positively and significantly correlated with age. The within-subject variablity was 30 % for interleukin-6,13% for its receptor, and 33.1% for the ratio interleukin-6 soluble receptor/interleukin-6. The between-subject variability was 43.7 % for interleukin-6, 22.2% for its receptor, and 60.2 % for the ratio interleukin-6 soluble receptor/interleukin-6. The indices of individuality (ratio within-subject variability/between-subject variability) were calculated and found to be 0.69, 0.60, and 0.55 for interleukin-6, its soluble receptor, and the ratio interleukin-6 soluble receptor/interleukin-6, respectively. The implications of these indices with the use of health-related reference values is discussed.

Concentrations of 34 biochemical constituents of sera were determined on 998 randomly selected urban school children and adolescents aged 8–18 years from Zagreb, Croatia. Reference intervals were obtained by using non-parametric methods to estimate 2.5 and 97.5 percentiles of distribution as upper and lower normal reference intervals, according to the IFCC recommendations. These were compared to reference intervals in the healthy adult population, aged 20–30 years from the same geographical area. Serum glucose, potassium, sodium, chloride, magnesium, iron, zinc, total serum proteins and electrophoretic fractions, and amylase, did not show age or sex differences; total serum bilirubin, total calcium, phosphate, high density lipoprotein cholesterol, total iron binding capacity, unsaturated iron binding capacity, copper, aspartate aminotransferase, alkaline phosphatase, cholinesterase, creatine kinase, and lactate dehydrogenase had higher reference intervals than the adult population. Urea, creatinine, urate, alanine aminotransferase, γ-glutamyltransferase, total cholesterol and low density lipoprotein-cholesterol, and triglycerides had lower reference intervals than the adult population.